?Structure-Guided Design of Broadly Neutralizing Lassa Virus BiSpecific Antibodies? ABSTRACT Lassa fever (LF) is an often-fatal viral hemorrhagic fever (VHF) that is endemic in West Africa where it causes significant social and economic disruption. The lack of an approved therapeutic or vaccine, recorded geographic expansion of rodent reservoirs, ease of procurement and weaponization of the virus, and the recent emergence of new Lassa virus (LASV) strains support recommendations for enhanced preparedness for LF. We isolated and characterized 113 human monoclonal antibodies (hMAbs), the first large panel of human antibodies against LASV described. We found that the most potent neutralizing hMAbs target quaternary epitopes that require both GP1 and GP2 subunits of each monomer in the glycoprotein complex (GPC) trimer. LASV is genetically diverse with four distinct lineages present in West Africa. Some hMAbs neutralized all 4 LASV lineages. Challenge of outbred guinea pigs (GP) in a model of lethal LF informed the down-selection of broadly neutralizing hMAbs (BNhMAbs) for studies in a nonhuman primate (NHP) model, Cynomolgus macaques. A combination of 3 BNhMAbs, each with broad neutralizing activity and recognition of distinct epitopes on the LASV GPC, rescued 100% of NHPs even after delay in the start of treatment to 8 days post-infection, a time when the animals displayed severe hematological and metabolic dysregulation. The 3 BNhMAb cocktail conferred 100% protection in NHP against lethal challenge with LASV strains from lineages II and IV. We now propose to utilize the structural information of BNhMAbs complexed with GPC to engineer human bi-specific antibodies (BsAbs) that span two highly protective epitopes, thereby reducing the number of molecules required to confer superior protection against LF. Preliminary results in LASV- challenged GP suggest that targeting quaternary neutralizing epitopes in the base of GPC with a bi-specific antibody results in superior protection, even at 10-fold lower doses than previoulsy tested for individual BNhMAbs (Preliminary Results). Our proposed project meets the strict requirements of RFA-AI-17-026 in that the LASV BsAbs are based on previously identified, well-characterized, candidate therapeutic hMAbs against an NIAID listed emerging pathogen, LASV. The project will address a particular interest of RFA-AI- 17-026 for immunotherapeutics that would ?enable prevention of infection or intoxication in the face of an immediate threat, protection of immunocompromised individuals, or post-exposure treatment to suppress infection and disease.? In Milestone 1 we will down-select BsAbs targeting base, middle, and cap neutralizing epitopes on LASV GPC. In Milestone 2 dose and dosing interval studies with mono and combination BsAb therapy in GP and NHP will be evaluated. In Milestone 3 Chemistry, Manufacturing and Control Data (CMC) will be established for leading BsAbs. In Milestone 4 we will perform Preclinical Pharmacology and Toxicology of BsAbs. At the conclusion of the proposed program we will complete preclinical evaluation of a first-in-class immunotherapeutic BsAb for the prophylactic and post-exposure treatment of LF. This application contains proprietary/priviledged information that Zalgen Labs and its subcontractors request not be released to persons outside the Government, except for the purposes of review and evaluations.